Abstract

Autophagy, a major degradative pathway for proteins and organelles, is essential for survival of mature neurons. Extensive autophagic-lysosomal pathology in Alzheimer's disease brain contributes to Alzheimer's disease pathogenesis, although the underlying mechanisms are not well understood. Here, we identified and characterized marked intraneuronal amyloid-β peptide/amyloid and lysosomal system pathology in the Alzheimer's disease mouse model TgCRND8 similar to that previously described in Alzheimer's disease brains. We further establish that the basis for these pathologies involves defective proteolytic clearance of neuronal autophagic substrates including amyloid-β peptide. To establish the pathogenic significance of these abnormalities, we enhanced lysosomal cathepsin activities and rates of autophagic protein turnover in TgCRND8 mice by genetically deleting cystatin B, an endogenous inhibitor of lysosomal cysteine proteases. Cystatin B deletion rescued autophagic-lysosomal pathology, reduced abnormal accumulations of amyloid-β peptide, ubiquitinated proteins and other autophagic substrates within autolysosomes/lysosomes and reduced intraneuronal amyloid-β peptide. The amelioration of lysosomal function in TgCRND8 markedly decreased extracellular amyloid deposition and total brain amyloid-β peptide 40 and 42 levels, and prevented the development of deficits of learning and memory in fear conditioning and olfactory habituation tests. Our findings support the pathogenic significance of autophagic-lysosomal dysfunction in Alzheimer's disease and indicate the potential value of restoring normal autophagy as an innovative therapeutic strategy for Alzheimer's disease.

Cystatin B deletion elevating lysosomal protein degradation. (A) Rates of proteolysis of long-lived proteins in fibroblasts from wild-type (WT) and CBKO (CB−/−) mice maintained in the presence (A1) or absence (A2) of serum. Where indicated a combination of ammonium chloride and leupeptin (leup) was added to inhibit proteolysis in the lysosomal compartment. Values are mean + SEM of three different experiments with triplicate samples. Mean differences between the untreated wild-type and CBKO (i.e. without lysosomal inhibitors) at each time point were analysed by two-tailed Student’s t-test. *P < 0.05. (B) Rates of proteolysis of long-lived proteins in primary neurons from wild-type and CBKO mice maintained in neurobasal medium supplemented with B27. Mean differences between wild-type and CBKO at each time point were analysed by two-tailed Student’s t-test. *P < 0.05.

Elimination of giant autolysosomes in the brains of CBKO/TgCRND8. Vibratome (A–C) or paraffin (D) brain sections from a group of wild-type, CBKO, TgCRND8 and CBKO/TgCRND8 mice (strains: 129S6 × 129X1 for the four genotypes) at 6 months of age were processed with antibodies to cathepsin D (RU2) (A), cathepsin B (B), cathepsin L (C) or Aβ (4G8) (D). All images are from the hippocampal CA1 sector showing reduction of giant autolysosomes—revealed by antibodies to either cathepsins or Aβ—in the CBKO/TgCRND8 compared to TgCRND8. The images in (A) are representative of the results from 9–13 mice per genotype, while those in (B–D) are from 3–4 mice per genotype. Arrowheads (A3) depict giant autolysosomes within the stratum radiatum. Scale bars: 20 µm (A–D). CatB = cathepsin B; CatD = cathespin D; CatL = cathepsin L; CRND8 = TgCRND8.